Exposure single-cell metabolomics mass spectrometry reveals HFPO-DA toxicity mechanisms.

Hexafluoropropylene oxide-dimer acid (HFPO-DA) is widely used in food packaging bags, surfactants, lubricants, etc. As a new substitute for the persistent organic pollutant perfluorooctanoic acid (PFOA), it has been proven to have potential health and environmental risks similar to other perfluorinated compounds. However, studies based on the average levels of many cells or tissues cannot accurately reflect the toxic mechanism of HFPO-DA. Here, we construct a high-throughput exposure single-cell metabolomics mass spectrometry analysis platform based on intact living-cell electrolaunching ionization MS analysis, optimize the detection conditions of the platform, and use it to investigate the effects of a wide range of HFPO-DA concentrations on the metabolism of mouse embryonic stem cells (E14TG2a cells). We evaluate the effect of HFPO-DA exposure on the viability of E14TG2a cells using the CCK8 method. The results showed that exposure to HFPO-DA at a concentration below 1 mmol L-1 within 24 hours had no significant effect on the viability of E14TG2a cells, while exposure above 1 mmol L-1 led to significant differences in cell viability. Comprehensive single-cell metabolic analysis revealed that even environmental concentrations of HFPO-DA exposure that did not affect cell viability could affect cellular metabolic changes, and the levels of some metabolites showed a non-monotonic dose-response relationship with HFPO-DA. HFPO-DA exposure interfered with metabolic pathways such as nitrogen metabolism, arginine biosynthesis, and arginine and proline metabolism, thereby affecting the homeostasis of basic biological metabolic processes such as nucleotide metabolism and amino acid metabolism in E14TG2a cells. This study indicates that although HFPO-DA has relatively low cytotoxicity, it can still interfere with the metabolic processes of E14TG2a cells, suggesting that the safety of HFPO-DA needs further evaluation.